The emergence of non-invasive live animal fluorescence imaging technology has opened new avenues for the development of cancer diagnosis and therapeutics. Fluorescence imaging probes with intense emission in the far-red/near-infrared (FR/NIR) region (>650 nm) are attracting increasing attention due to their ability to overcome the interferences of optical absorption, light scattering, and auto-fluorescence of biological media.
To date, a large variety of materials, including organic dyes, fluorescent proteins, and inorganic quantum dots (QDs), have been extensively studied for the purpose of FR/NIR fluorescence imaging. Organic dyes and fluorescent proteins, however, suffer from limited molar absorptivity and low photobleaching thresholds, while inorganic QDs are highly cytotoxic in an oxidative environment (A. M. Derfus et al., Nano Lett. 2004, 4, 11). This has greatly limited the application of organic dyes, fluorescent proteins, and QDs for in vitro and in vivo imaging.
Fluorescent nanoparticles, such as organic fluorophore-loaded nanoparticles, have recently emerged as a new generation of nanoprobes for bioimaging. They exhibit advantages such as synthetic versatility, low cytotoxicity, high photostability, and facile surface functionalization for specific targeting. For practical applications, brightly emissive nanoparticles are desirable for high contrast imaging. Ideally, the brightness of fluorophore-doped nanoparticles should be proportional to the number of encapsulated dye molecules. However, at high loading contents, π-conjugated fluorophores are prone to aggregate. The aggregate formation often quenches light emission, a common photophysical phenomenon known as aggregation caused quenching (ACQ). The ACQ effect has prevented the fabrication of nanoparticles with high degrees of brightness. Effort has been made to amplify the fluorescence of dyes with the ACQ property (U.S. Pat. No. 7,883,900). However, even after amplification, the fluorescence signal has been only mildly enhanced.
Most organic fluorophores including ethidium bromide (U.S. Pat. Nos. 4,729,947, 5,346,603, 6,143,151, and 6,143,153), Nile red (U.S. Pat. Nos. 6,897,297 and 6,465,208), fluorescamine (U.S. Pat. No. 4,203,967), o-phthaldialdehyde (U.S. Pat. Nos. 6,969,615 and 6,607,918), and Cyanine dyes (U.S. Pat. Nos. 5,627,027 and 5,410,030) are emissive only in their solution state. Emission is significantly quenched or completely quenched in aggregation states (i.e., high dye concentration state, film state, solid powder state, etc.). Therefore, the loading concentration of dyes in polymer particles can only reach moderate levels, resulting in limited achievable fluorescence intensity. Therefore, the practical applications of organic fluorophore-doped nanoparticles for in vitro and in vivo bioimaging are considerably limited.
Accordingly, there is a great need for the development of fluorescent bioprobes with high biological compatibility, strong photobleaching resistance, and efficient light emission for use in in vitro and in vivo imaging, particularly live animal imaging.